The reliability of immunoassays to detect autoantibodies in patients with myositis is dependent on autoantibody specificity

Abstract

Objectives In order to address the reliability of commercial assays to identify myositis-specific and -associated autoantibodies, we aimed to compare the results of two commercial immunoassays with the results obtained by protein immunoprecipitation. Methods Autoantibody status was determined using radio-labelled protein immunoprecipitation for patients referred to our laboratory for myositis autoantibody characterization. For each autoantibody of interest, the sera from 25 different patients were analysed by line blot (Euroline Myositis Antigen Profile 4, EuroImmun, Lübeck, Germany) and dot blot (D-Tek BlueDiver, Diagnostic Technology, Belrose, NSW, Australia). Sera from 134 adult healthy controls were analysed. Results Overall commercial assays performed reasonably well, with high agreement (Cohen’s κ >0.8). Notable exceptions were the detection of rarer anti-synthetases with κ < 0.2 and detection of anti-TIF1γ, where κ was 0.70 for the line blot and 0.31 for dot blot. Further analysis suggested that the proportion of patients with anti-TIF1γ may recognize a conformational epitope, limiting the ability of blotting-based assays that utilize denatured antigen to detect this clinically important autoantibody. A false-positive result occurred in 13.7% of samples analysed by line blot and 12.1% analysed by dot blot. Conclusion The assays analysed do not perform well for all myositis-specific and -associated autoantibodies and overall false positives are relatively common. It is crucial that clinicians are aware of the limitations of the methods used by their local laboratory. Results must be interpreted within the clinical context and immunoprecipitation should still be considered in selected cases, such as apparently autoantibody-negative patients where anti-synthetase syndrome is suspected.

Anti-nuclear matrix protein 2 antibody-positive idiopathic inflammatory myopathies represent extensive myositis without dermatomyositis-specific rash

“…In this study, the ANA titer was low (<1:80) in half of the patients with anti-NXP2 Antibody. This result suggested that the anti-NXP2 antibody titer was relatively low, which may explained that the sensitivity of a commercial blot assay kit for detecting anti-NXP2 antibody was low in a previous report (15).…”

Identification and prediction of novel classes of long-term disease trajectories for patients with juvenile dermatomyositis using growth mixture models

“…While it would have been useful to identify groups with similar muscle disease activities over time, there were too many missing CMAS values to do so. Although 96% of our autoantibody data were generated using the same method, inconsistent testing methods are a challenge for combining data [ 33 , 34 ]. Future work could investigate how medication exposure both influences and is influenced by disease trajectories.…”

Comparison of Lesional Juvenile Myositis and Lupus Skin Reveals Overlapping Yet Unique Disease Pathophysiology

“…These clinically available MSA testing methodologies utilize differing techniques for MSA detection, with the OMRF profile determined predominantly by immunoprecipitation and immunoblotting and the Myomarker Panel 3 by enzyme immunoassay (EIA). While a direct comparison of test performance characteristics between these 2 methodologies has not been published for reference, it has been noted that EIA methodology has a lower sensitivity for detection of some autoantibodies, such as anti-transcription intermediary factor 1γ (anti-TIF1γ), while also potentially leading to more false-positive results (16)(17)(18)(19).…”